EXTRINSIC COTTON EFFECTS I N RETINALDEHYDE SCHIFF BASES
Ross, D. L., and Jirgensons, B. (1968), J. Biol. Chem. 243, 2829. Sarma, V. R., Silverton, E. W., Davies, D . R., and Terry, W. D. (1971), J . Biol. Chem. 246, 3753. Schiffer, M., Hardman, K. D., Wood, M. K., Edmundson, A. B., Hook, M . E., and Ely, K . R . (1970), J. Biol. Chem. 245,728. Schramm, H. J. (1971), Hoppe-Seyler’s 2.Physiol. Chem. 352, 1134. Seon, B.-K., Roholt, 0. A., and Pressman, D. (1969), Biochim. Biophys. Acta 194,397. Seon, B.-K., Roholt, 0. A., and Pressman, D. (1971), J. Biol. Chem. 246, 887. Shotton, D. M., and Watson, H. C. (1970), Phil. Truns. Roy. SOC. London, Ser. B 257,111. Singer, S. J., and Doolittle, R. F. (1966), Science 153, 13. Singer, S. J., Martin, N., and Thorpe, N. 0. (1971), Ann. N. Y . Acad. Sci. 190,342. Solomon, A., and McLaughlin, C. L. (1969), J . Biol. Chem.
244,3393. Solomon, A., McLaughlin, C. L., Wei, C. H., and Einstein, J. R. (1970), J . Biol. Chem. 245,5289. Stevenson, G .T. (1973), Biochem. J.(in press). Stevenson, G . T., and Dorrington, K. J. (1970), Biochem. J. 118, 703. Strosberg, A. D., Fraser, K. J., Margolies, M. N., and Haber, E. (1972), Biochemistry 11,4978. Thorpe, N. O., and Singer, S. J. (1969), Biochemistry 8,4523. Titani, K., Whitley, E., Jr., and Putnam, F. W. (1966), Science 152,1513. Tomasi, T. B., and Grey, H . M. (1972), Progr. Allergy 16, 81. Wikler, M., and Putnam, F. W. (1970), J. Biol. Chem. 245, 4488. W u , T . T . , a n d K a b a t , E. A. (1970),J. Exp. Med. 132,211. Wyckoff, H. W., Doscher, M., Tsernoglou, D., Inagami, T., Johnson, L. N., Hardman, K. D., Allewell, N. M., Kelly, D. M., and Richards, F. M. (1967), J. Mo/. Biol. 27, 563.
Extrinsic Cotton Effects in Retinaldehyde Schiff’s Basest Edward M. Johnston and Robert Zand*
ABSTRACT : Schiff’s bases have been synthesized from all-transretinaldehyde and (S)-a-phenylethylamine, S-a-(1-naphthyl)ethylamine, R-(+)-2,2’-dimethyl-6,6’-diaminobiphenyl,and poly-L-lysine. The Schiff‘s base of 11-cis-retinaldehyde with (S)-a-(1-naphthy1)ethylaminewas also prepared. The absorption and circular dichroic spectra were measured for the parent compound and the protonated forms over the wavelength region of 190-700 nm. Extrinsic Cotton effects were observed in the region of the major retinaldehyde absorption
T
he visual pigment rhodopsin exhibits a broad circular dichroic (CD) band in the region of 500 nm where the ll-cisretinaldehyde component of rhodopsin has an absorption maximum. all-trans- and 11-cis-retinaldehyde are optically inactive molecules and the mechanism for the induction of the optical activity as well as the significance of the problem relative to the structure of rhodopsin is an unsolved problem. In 1966, the C D spectrum of bovine rhodopsin was reported (Crescitelli er a/., 1966). The initial explanation that was considered and then rejected by these workers was that the optical activity arose from an extrinsic mechanism. The explanation advanced for the induction of optical activity into the retinaldehyde chromophore was based on the hypothesis that the binding of 11-cis-retinaldehyde to opsin was ac~ _ _ _ _ _ _ _ _ _ _
t From
.___
the Biophysics Research Division, Institute of Science and Technology, and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48105. Receiced Februarj> 9, 1973. This work includes material from a thesis submitted by E. M. J . in partial fulfillment of the requirements for the degree of Doctor of Philosophy. A preliminary report of this work has been published (Johnston and Zand, 1972). Supported in part by National Institutes of Health Grants NB-05036 GM-1355 and GM-14035.
band centered at about 360 nm. Protonation of the Schiff’s base caused the absorption and circular dichroism band to shift to about 410 nm. The Schiff’s base, W-all-trans-retinylidene-L-lysine and N-all-trans-retinylidene-L-dipalmitoylphosphatidylethanolamine did not exhibit extrinsic Cotton effects in the region of retinaldehyde absorption. The results are pertinent to the origins of the observed Cotton effect in the visible wavelength region of rhodopsin.
companied by a twisting of the aldehyde into an asymmetric conformation. The twisted chromophore concept was developed further (Mommaerts, 1969) by suggesting that l l-cisretinaldehyde could exist in two enantiomeric forms (Figure 1). When the 11-cis-retinaldehyde is bound to opsin one enantiomer was thought to be preferentially stabilized and “frozen in” thereby providing the asymmetry necessary for optical activity. The concept of intrinsic optical activity in the retinaldehyde portion of rhodopsin has recently been treated more extensively (Honig et ul., 1973). The extrinsic mechanism was reintroduced by Kito et a/. (1968) to explain the observed Cotton effects in squid rhodopsin. This approach was developed further by Johnston and Zand (1972) and Waggoner and Stryer (1971). In this mechanism the optical activity may arise through a Kirkwood-type coupled oscillator mechanism (Kirkwood, 1937) so that the retinaldehyde transitions may become optically active by coupling with the transitions of nearby aromatic side chains. The present study was undertaken with the view that spectral studies of Schiff’s bases (Table I) derived from retinaldehyde and appropriate optically active amines would provide information that would help to establish the mechaBIOCHEMISTRY,
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TABLE
Ac
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Experimental Data: Dissymmetry Factors (g
-=
6). .~
Absorption Amine Moiety __
\ S
-
‘1,
~-
Solvent
c L)
I\
(nm) S (nm) K X 10a ~-
-
A. Unprotonated Schiff’s Bases between the Named Amine and all-truns-Retinal (S)-NEA“ isooctane 360 354 -,-I 1 5 302 0 282 -12 7 (S)-NEA CHCI j 370 370 12 I
(S)-PEA
isooctane
350
(S)-PEA
C‘HCI,
369
DMAB
Isooctane
371
4 5 0
-20 7 13 3 0 -1.1 5
i 52 7 0
--44 4 i 11 3
dehyde. nism by which optical activity was transmitted into the 1l-cisretinaldehyde chromophore. Additional information relating to the structure of opsin might also be provided by such spectral studies.
B. Unprotonated SchifYs Base between the Named Amine and 1 I-ctA-Retinal (S)-NEA Isooctane 350 350 4 (1 (not a peak, see spectrum)
1 : A representation of the two conformers of 1 l-cis-retinal-
all-trans-Retinaldehyde was obtained from Eastman Kodak Co. Quantities of 11-cis-retinaldehyde were gifts from Paul Brown of Harvard University and from Hoffman-LaRoche Co., Nutley, N . J. The retinaldehydes were stored in the dark a t -40” until needed. The amines, (-)-(S)-a-phenylethylamine and ( -)-(S)-a-(1-naphthyl)ethylamine, were obtained from Norse Laboratories, Santa Barbara, Calif. Dipalmitoylphosphatidylethanolamine was obtained from Mann Research Laboratories of New York. The (+)-(R)-2,2’-dimethy1-6,6’diaminobiphenyl was synthesized and resolved by the procedure reported by Meisenheimer and Horing (1927). PolyL-lysine hydrobromide, mol wt 92,000, was obtained frorn Pilot Chemicals Inc., Watertown, Mass. The hydrochloride salt was prepared by exhaustive dialysis of the hydrobromide against 0.1 N HCI, followed by lyophilization. Absorption spectra were recorded on a Cary 15 spectrophotometer. CD and O R D spectra were recorded o n a Jasco ORDjCD instrument incorporating the SS-20 modification. (S)-N-aN-truns-Retinylidene-a-(l-naphthyl)ethylamine. To 1 ml of dry chloroform was added 14.18 mg (0.050 mM) of alltrans-retinaldehyde and 11.18 mg (0.065 mM) of (S)-a-(1naphthy1)ethylamine. The solution was allowed to stand overnight in the dark at room temperature. The reaction mixture was diluted 1515:l with isooctane and absorption and CD spectra were recorded. Completion of the reaction was verified by diluting some of the original chloroform solution with more chloroform and bubbling in gaseous HCI. The 370-nm peak disappeared completely and a new peak formed B I O C H E M I S T R YV,O L . 1 2 ,
0 -- I O h
7
C . Protonated Schiff‘s Bases between the Named Aminc and a//-frons-Retinal
Materials and Methods
4632
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0 10 9
--
Poly-L-lysine pH 11 46, (Y helix)
FIGURE
5 9 (v, v) methanol water
315 293 354 293 254 376 3’0 262 400 374 346 375 350 330
NO.
23, 1 9 7 3
(S)-NEA (S)-PEA
Poly-L-lysine (pH 2 0. U EH) (S)-NEA
CHC‘I j CHCI
7 % (v v ) methanol water
x
,\
468 464
460 490 410 273 350 406 370
3Y4
D. Amines alone Isooctane 282
223
(S)-PEA (R)-DMAB
Isooctane Isooctane
258 291
PE
H FA5
218
__
. _
-
293 286 264, 233 227 208 200 262 300 2 94 284 210
g
x
10,
-7 5 ii7 (J
-2 8 -9 0 0 i X h
-3 2 -3 2 0 - 1 8
0 -8 0 t56 t58 0 -91 LI9
t: 8 1
3
7
~
Abbreviations used are: NEA, (S)-(- )-w(l-naphthyl)ethylamme; PEA, (S)-(-))-a-phenylethylamine, DMAB, (R)-(+)-2,2 ’-d1methyl-6,6’-d1amlnobiphenyl, PE. L-dipalmitoylphosphatidylethanolamine; HFAS, hrxafluoroacetone sesquihydrate.
EXTRINSIC COTTON
EFFECTS IN
RETINALDEHYDE SCHIFF BASES
a t 468 nm. The presence of unreacted retinal would have been revealed by a peak a t 390 nm. Protonated (S)-N-all-rrans-Rerinylidene-a-(I-naphthyl)erhylamine. The reaction was carried out as described for the preparation of the unprotonated form. It was then diluted with chloroform to a concentration suitable for spectral measurements. To this chloroform solution was added several drops of chloroform previously treated with dry gaseous HCl. The resulting solution was used immediately t o obtain absorption and CD spectra. (S)-N-all-trans-Retinylidene-a-phenq'lethylamine. To 1 ml a W of chloroform was added 14.27 mg (0.050 mM) of all-trans'-I retinaldehyde and 9.40 mg (0.078 mM) of (S)-a-phenylethylW amine. The solution was allowed t o react overnight in the dark a t room temperature. The reaction mixture was diluted 758 :1 with isooctane and the resulting solution was used im-6 , 1 , , ~ , , , , ~ , , , , mediately for absorption and CD spectra. 300 400 500, Protonated (S)-N-all-trans-Rerinylidene-a-phenylerhylan~ine. Wavalem#h (nm) The reaction was carried out as for the unprotonated form. The solution was then diluted appropriately with chloroform. FIGURE 2: (a) The absorption spectra of the protonated and unprotonated forms of (S)-N-ull-tm/is-retinrlidene a-(I-naphthy1)ethylTo this chloroform solution was now added 1 or 2 drops of amine in isooctane solution. (b) The CD spectra of the protonated HC1-treated chloroform and the absorption and C D spectra and unprotonated Schiff's base. These spectra have been corrected were recorded. by subtracting out the CD contribution from 30% excess amine. (R)-N-all-trans-Retinylidene-2,2'-dimethyl-6,6'-diaminobiphenyl. To 1 ml of carbon tetrachloride was added 14.21 mg utilized for absorption and CD spectra and to check for com(0.050 mM) of all-trans-retinal and 21.09 mg (0.099 mM) of pleteness of reaction as previously indicated. (R)-2,2'dimethyl-6,6'-diaminobiphenyl.The mixture was alN'-all-trans-Rerinylidene-pol~~-L-lysine. To a solution of lowed to react overnight in the dark at room temperature. 6.17 mg (0.037 mM lysine residues) of poly-L-lysine in 0.5 ml It was then diluted 2800:l with isooctane. Addition of several of distilled water and 40 p1 of NaOH was added 0.5 ml of drops of HCI-treated chloroform shifted the absorption maximethanol containing 0.1945 mg of all-trans-retinaldehyde mum from 386 to 495 nm. N o detectable peak was present (0.00068 mM). The mole ratio of lysine residues to retinal between 350 and 400 nm. molecules was thus 54 :1. The mixture was allowed t o react (S)-N-1I-cis-Rerinylidene-a-(l-naphrhyl)ethyIarnine. To 1 overnight in the dark at room temperature. An aliquot of this ml of chloroform was added 7.16 mg (0.025 mM) of 11-cissolution was diluted by the addition of 7.4volumes of water retinaldehyde and 5.35 mg (0.031 mhi) of (S)-a-(1-naphthyl)and the p H was adjusted to 11.46by the addition of 1 N sodium ethylamine. The mixture was allowed to react for 1 hr in the hydroxide. Absorption and C D spectra were recorded on this dark at room temperature and then diluted 500 :1 with isosolution. To convert the poly-L-lysine to the p conformation octane. Complete reaction was determined by the addition the sample was heated to 50" by passing an ethylene glycolof HC1-saturated chloroform. This resulted in the disappearwater mixture from a circulating bath through the water ance of the 366-nm peak and the appearance of a new peak jacket of the cell. After about 20 min no further change in a t 467 nm. the CD at 224 n m was observed and the complete C D specN - all- rrans- Retinylidene-L-dipalmiroylphospharidylethanoltrum was then recorded. amine. Chloroform and absolute ethanol were dried with Protonated N'-all-trans-Rerinylidene-poly-L-l~~sine.The Linde 3A molecule sieve and then mixed to form a 2 :1 (v/v) Schiff's base was prepared as described for the unprotonated chloroform-ethanol solvent mixture. To 10 ml of 2 :1 chloroa-helical form. Next, the p H was adjusted to 2.0 by the addiform-ethanol was added 8.18 mg (0.029 mM) of all-transtion of concentrated HC1. Absorption and C D spectra were retinaldehyde. To 1.99 ml of 2 : 1 chloroform-ethanol was added 4.82 mg (0.0073 mM) of L-dipalmitoylphosphatidyl- obtained on the resulting solution. ethanolamine. To the phosphatidylethanolamine solution Nas now added 1 ml of the retinaldehyde stock solution conResults and Discussion taining 0.0029 mM retinaldehyde and 10 pl of triethylamine t o give a final volume of 3 ml. The mixture was allowed to The C D spectrum of the Schiff's base formed from allreact overnight in the dark at room temperature. An aliquot trans-retinaldehyde and (S)-a-(1-naphthy1)ethylamim is shown of this solution was removed, diluted with 14 volumes of in Figure 2.Also shown is the CD spectrum for the protonated chloroform, and the absorption spectrum was obtained. Comform of this compound and for comparison the C D and abpleteness of reaction was ascertained by the absence of a band sorption spectra of the free amine shown in Figure 3. The a t 390 nm and by the addition of acidified chloroform. presence of a Cotton effect at 354 nm with a rotational strength N'-all-trans-Retinylidene-~-l.~sine. The procedure utilized of +0.27 D B M is noteworthy. all-trans-Retinal absorbs at was that of Akhtar et al. (1968). To 50.08 mg (0.18 mM) of 368 nm in isooctane and is optically inactive. The amine by all-trans-retinaldehyde in 10 ml of methanol was added 1 ml itself has no circular dichroism in the 360-nm region; hence of a distilled water solution containing 35.74 mg (0.20 mM) the observed band is a consequence of the formation of the of L-lysine monohydrochloride and 7.3 mg of triethylamine. Schiff's base. This Cotton effect at 354 nm can be assigned to The mixture was allowed to react in the dark at room temthe retinylidene band which occurs at 360 nm in the Schiff's perature for 6 hr, after which time a sample was withdrawn base and at 368 nm in free all-trans-retinaldehyde. Moving and diluted 50:l with methanol. The diluted solution was through the CD spectrum we observe a negative Cotton effect
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'
'
'
'
3b0 '
AND
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WAVELENGTH (nm)
3: The absorption and circular dichroism spectra of ( S ) - a (1-napthy1)ethylaminei n isooctane solution. FIGURE
at 282 nm having a rotational strength of -0.116 DBM. This band at 282 nm is about 20 times larger than that of the free amine, although it coincides with the 'La transition of the amine. The origin of this band can be shown to arise from the interaction of the 282-nm transition of the naphthyl group with the 360-nm transition of the retinylidene group. The interaction is such that the 282-nm transition acquires a negative value for R and the 360-nm transition acquires a positive value for R. Below 250 nm, the free amine has strong optical activity and must be subtracted out from the C D spectrum of the Schiff's base to find the new Cotton effects induced by Schiff's base formation. When this is done, induced peaks are found at 246, 227, and 208 nm. The R.14,j = -0.027 DBM. R?,; = ;-0.062 DBM, and R:?., = -0.120 DBM. The band at 246 nm corresponds to no obvious absorption or CD band in the free naphthylethylamine. The Cotton effect at 227 nni very likely corresponds to the 224 nm IBI, transition that is present in the parent amine.
6.
4.
2.
c
w 0
1
w"
.z
-4.
-6.
F I G U R E 4:The absorption and CD spectra of (.S)-.V-l 1-ci.s-retinyIii n isooctane solution. The solution dene-a-( I-naplith~l)eth~lamine contains a 23 ",;excess of amine over the Schiff's base.
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5 : The absorption and CD hpectra ~ ~ ( S ) - , ~ ~ - ' ~ ~ / - / ~ ~ ~ f i \ - r e ~ i i i ~ I cleiie-a-plienyletli4lainine i n isooctane solution. T!ie solution contains a 5 5 " ; K X C ~ S Sof amine over the Schiff's base. FIGUKL:
Experimentall) it was not possible to measure spectra below 200 nm, but additional information may be obtained about that spectral region based on the following argument. Klevens and Platt (1949) repcrted that the 'B, transition of naphthalene was located at 167 n m . If the (S)-a-(1-naphthyl)ethylamine alho absorbs at this wavelength and if this is the only other optically active transition in the spectrum. then according to the Kuhn sum rule (Kuhn, 1930), it must have R,,!, = -0.067 DBM. The use of the Kuhn s ~ i mrule in the absence of more definitive assignments of the possible transitions below 200 nm, places the above calculation on I e s firm ground than normally desirable. However: in the absence of these firm assignments, it is legitimate to carry out the calculation using the Klekens and Platt assignment in order to indicate where the remaining rotational strength of the system is located and potentially assignable. The formation of the Schiff's base from (S)-a-(l-naphthyljethylamine and 11-eis-retinaldehyde gives rise to a C D spectrum (Figure 4) that is \ t r y diferent from the one exhibited b y the Schiff's base of the all-trans isomer. Whereas the alltrans compound has a positive Cotton effect at 354 nm, the 11-cis compound exhibits a C D spectrum that is rather amorphous in the region of 300400 nm with no defined Cotton etfect a t 351 nni. This is clear evidence that no isomerization of the 1 l-c,i.s to rill-tr~ris-retinylidene group occurred during the scan. An explanation for the amorphous appearance of the CD spectrum based on cis -+ trans isomerization beenis \inlikely since such an isomerization would convert a sufficient fraction of the molecules to the all-trans form to produce a Cotton elTeet at 354 nm. After eight spectra were traced of the same bolution a new peak did appear at 380 nm. A well-detined positive peak is present at 280 nm and a negative peak at 218 nm. The lack of a distinct Cotton efkct a t 354 nm in the 11-cis compound may be a reflection of a lack of conformational rigidity that is present in huch a simple analog of rhodopsin. The CPK model suggests that fret: rotation is possible at the C , . - C t Rsingle bond of the 11-cis isomer. Studies by Nash
EXTRINSIC COTTON EFFECTS IN RETINALDEHYDE SCHIFF BASES
-. k
X
c
z
L
w
'>
2
4
\u I1
Y
v
Y
250
400
4 0
o w
a
Y W
-2
0 W e n g t h (nm)
6 : The absorption and CD spectra of the protonated form of (S)-N-nll-tra,is-retinylidene-a-phenylethylamine in chloroform solution. The solution contains a 5 5 % excess of amine hydrochloride over the Schiff's base hydrochloride. FIGURE
(1969), Honig et al. (1971), and Honig and Karplus (1971) have been interpreted by them as showing that the S-cis and S-trans conformers about the CI2-Cla bond are probably in thermal equilibrium in solution at room temperature. This would imply the presence of a very low-energy barrier to rotation under these conditions and supports our observations made with C P K models. If this rotation is in fact free, the observed CD spectrum would be a n average over a wide range of relative orientations for the retinylidene transition dipole moment and the naphthyl transition dipole moment so that the observed CD spectrum would exhibit a n amorphous character. Protonation of the 11-cis Schiff's base gave rise to variable and nonreproducible CD spectra. In Figures 5 and 6 the CD and absorption spectra for the unprotonated and protonated are forms of (S)-N-all-trans-retinylidene-a-phenylethylamine shown. The Schiff's base derived from (S)-phenylethylamine and all-trans-retinaldehyde shows the typical major band at 359 n m in the absorption spectrum and a 354-nm band in the C D spectrum, The 254-nm negative Cotton effect is assigned to the 'Lb transition of the phenyl moiety. The 'Lb negative Cotton effect induces an equal positive Cotton effect in the retinal. The phenyl group also acquires a second induced positive band a t 219 n m that is not assigned a t this time. It may be the 1L, band of the phenyl group which would be expected near 203 nm. The degenerate lB, and 'Bb bands are reported by Salem (1966) to lie in the region of 180 nm much too far from 219 nm to be the likely transitions responsible. When this Schiff's base is protonated the A, in the in absorption spectrum is shifted to 464 nm while the, , ,A the CD spectrum is shifted to 490 nm. For the Schiff's base derived from all-trans-retinaldehyde and (R)-2,2'-dimethyl-6,6'-diaminobiphenylin which it is possible that 2 mol of aldehyde are coupled t o 1 mol of amine, we observe the induction of a double Cotton effect (Figure 7). There is a crossover in the C D a t 374 nm which is close t o the absorption maximum of 371 nm, and this is flanked by a positive Cotton effect at 400 nm and a negative Cotton effect a t 346 nm. For the macromolecular system N'-all-trans-retinylidenepoly-L-lysine, in which the ratio of retinaldehyde molecules to L-lysine residues was kept at about 1 : 54, a n interesting biphasic spectrum was found as shown in Figure 8. In the a-helical conformation a peak is present a t 375 nm, a crossover at 350 nm, and a trough at 330 nm. When the a-helical
U
E
VI
0
2
-
0 I-
u
z
-c X Y
'-16
I SOOCTANE
NANOMETERS
7 : The absorption spectra of nl/-tra,is-retinaldehyde-(R)(+)-2,2'-dimethyl-6,6'-diaminobiphenyl and the Schiff's base formed from them. The CD spectrum IS also shown. All spectra are in isooctane solution and those of the Schiff's base contain a 99%, excess of the free amine. FIGURE
form is converted to the /3 form by heating, the circular dichroism was not abolished. The C D curves resemble the original a-helical spectrum decreased in intensity by 4 0 z . When the pH of the solution is adjusted to 2.0, the poly-L-lysine converts to a form designated as unordered-extended helix (Dearborn and Wetlaufer, 1970), the C D spectrum is markedly changed (Figure 9). The biphasic form is retained but now a negative Cotton effect is present a t 450 nm, a crossover occurs at 406 nm, and a positive Cotton effect is now centered at 370 nm.
- -3. I
220
1
1
,
1
,
I
,
,
4 00
300 Wavelength (nm)
FIGURE 8 : The absorption and CD spectra of N'-ull-truns-retinylidene-poly-L-lysine in 5.9 (v/v) methanol-water solution. The pH is 11.46, the poly-L-lysine conformation is a helical and there is a 60 : 1 molar excess of lysine residues over retinylidene residues.
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Wovekngth (nm) Fjc;uRE 9 : The absorption and CD spectra of the protonated form of N'-n//-i,iiiJ.s-retjn).lidene-polq.-L-l~sine in 7 y ; (v/v) methanol-water solution. The pH is 2.0, the poly-L-lysine conformation is unorderedextended helix. There is also a 60 : 1 molar excess of lysine residues
over retinylidene residues.
The appearance of biphasic Cotton effects in the spectra of N-nl/-truns-retinylidene-2,2 '-dimethyl-6,6 '-diaminobiphen yl and N'-all-truns-retinylidene-poly-L-lysineunprotonated and protonated might be explicable on the basis of retinal-retinal interactions. The amine component of these Schiff's bases have two or more amino groups available for reaction with molecules of retinaldehyde. If two retinaldehyde molecules react at adjacent sites that are asymmetrically arranged, they can induce optical activity in each other. Alternatively if two retinaldehydes interact intermolecularly they can also induce optical activity in each other in these systems. If one of the two retinaldehyde acquires a positive rotational strength, the other must acquire a negative one in order to keep the sum of the rotational strengths zero for the whole spectrum. In general, these two Cotton effects will occur a t slightly different wavelengths, so they will produce a peak-trough spectrum like that observed rather than cancelling out. It is possible that the circular dichroism induced in each retinaldehyde is greater than would appear from the spectrum. Such a situation is possible when two approximately Gaussian curves of opposite sign are slightly offset and then added. Under this circumstance they will tend to cancel and produce a crossover in the region where they overlap while their maxima will be somewhat diminished in intensity. The positive and negative Cotton effects a t 400 and 376 nm, respectively, are separated by a crossover point at 374 nm. This value is sufficiently close to the absorption maximum of 371 nm as to make the tentative explanation attractive. In the N'-N/I-trans-retinylidene-poly-L-lysineC D spectra we again observe a crossover centered near the peak of the absorption maximum flanked by a biphasic Cotton effect. Three explanations can be offered for the biphasic effect in these systems. One possibility is that the retinaldehydes are binding to adjacent +amino groups and are held in an asymmetric configuration by the helical structure of the polymer backbone. A second possibility is that the CD is induced merely by thc asymmetric electric field due to the helix. The third explanation that would also account for the observed C D spectrum is that the polymer, though predominantly helical, may also contain segments that are folded. Thus a retinal attached to residue X may be adjacent to a retinal at residue Y even though in the completely extended form these residues would be separated by appreciable distances. Since the cir-
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AND
Z,\ND
cular dichroism is not abolished when the a-helical structure is converted to the /3 structure. it would appear that adjacent retinal sites remain unchanged in the cy and 8 transition. The inversion of Cotton effects that occurs when the pH is adjusted to 2.0 eludes a definitive explanation at this tinic. The backbone conformation at this pH is the so called tinorderedextended helix conformation. The Schiff's base N-oil-trans-retinylidene-L-dipalmitoylphosphatidylethanolamine showed no extrinsic Cotton effect in the region of the retinaldehyde absorption maximum. That the Schiff's base was formed could be readily demonstrated by the red shift of the main absorption peak on protonation, A comparable situation was present in the C D spectrum of N'-u//-rv~ns-retinylidene-L-lysine. No optical activity could be demonstrated in the region of 380 mi. The absence of extrinsic Cotton elTects in the compounds N'-retinylidene-L-lysine and N-retinylidene-L-dipalmitoylphosphatidylethanolamine is perhaps not surprising. The r-lysifie component of N-retiny lidene-L-lysine and the L-phosphatidylethanolamine group ha\ e no strong transition that could interact with the retinylidene to induce optical activity. Moreover. since the retinal is attached to the e-amino group in one instance and the ethanolamine group in the other. i t is quite remote from the carbonyl group of these molecules. which is the only double-bonded unit in the molecules. The single bonds in the side chain of lysine and ethanolamine confer conformational flexibility on the whole system, bending to average out any small C D that might have been induced. The results of this study using Schiff's bases of retinaldehyde and small, optically active aromatic amines indicate that the induction of optical actibity in the retinaldehyde moiety does not require the imposition of conformational asymmetry on the backbone of the retinaldehyde. as has been proposed for rhodopsin. References Akhtar, M., Blosse, P. T.. and Dewhurst, P. B. (1968): Biodiem. J . 110, 693. Crescitelli, F., Mommaerts, W. F. H. M.. and Shah. T. I. (1966). Proc. Nut. Acud. Sri. L'. S . 56, 1729. Dearborn, D. G., and Wetlaufer. D. B. (1970). Biociie/ii. BiopliJ.s. Res. Coniririm.39, 314. Honig, B.. Hudson. B.. Sykes, B.. and Karplus, M . (l971), Pror. Nut. Arad. Sci. c'. S 6 8 , 1289. Honig. B.. Kahn. P., and Ebrey, T. G . (1973). Bioc/wnii.strj* 12. 1637. Honig, B.. and Karplus, M. (1971), Nuturr(London) 22Y, 5%. Johnston, E. M., and Zand, R . (1972). Biorliem. Bioplq's. Rrs. co/llll?l/n.47, 7 13. Kirkwood. J. G. (1937). J . Cheni. Phj..r.5,47Y. Kito, Y . , Azuma, M., and Maeda, Y . (1968), Liiochi/u. Bioplij,.s.Actci 1.54, 352. Klevens, H. B . , and Platt, J. R. (1Y4Y),J . Cheni. P/IJ,.s. 17, 470. Kuhn, W. (1930). Trans. Furudu>,Soc.26,293. Meisenheimer, J.. andHoring, M. (1927), Ber. 60B. 1425. Mommaerts, W. F. H. M. (1969), in The Retina: Morphology, Function and Clinical Characteristics, Straatsma, B. R., Hall, M. O., Allen, R . A., and Crescitelli, F.. Ed.. Los Angeles. Calif., University of California Press, p 225. Nash, H . A. (1969),J. Theor. Biol. 22, 214. Salem. L. (1966), The Molecular Orbital Theory of ('onjugated Systems, New York, N. Y., W. A. Benjamin. Waggoner. A. S.,and Stryer, L. (1971), Biocheniistrj, 10, 3250.
